Technical Abstract:
Enteric virus-contaminated molluscan shellfish continue to cause illness throughout the world. The advent of molecular biological methods has enhanced the ability to detect many of these viruses. Prior to analysis, viruses must be extracted from shellfish tissues and concentrated. We developed a method to extract and concentrate the RNA from hepatitis A and hepatitis E viruses and from noroviruses isolated from oysters and clams. The concentrated RNAs are then assayed by reverse-transcription polymerase chain reaction (RT-PCR) and the presence of PCR product signals virus presence. The RT-PCR method works well on the hepatitis viruses which have conserved genomes, but fails to amplify many of the noroviruses which have highly diverse genomes. We developed real-time RT-PCR (rt RT-PCR) procedures to detect hepatitis A and E viruses. We also developed methods to detect a wide array of genogroup I and II noroviruses using broadly reactive primers and SYBR green detection. Product verification is rapid and simple, and is accomplished by means of first derivative melt curve data. The extraction and assay procedures allow for the detection and semi-quantitation of noroviruses from at least thirteen different genetic clusters. This rt RT-PCR technique has been used on stool preparations in epidemiological investigations to identify the sources of norovirus transmission and is under evaluation for use on shellfish extracts.